The present invention relates to the analytical chemistry arts. It finds particular application in conjunction with the titrimetric analysis of microliter size samples. It also finds application in conjunction with instrumentation for electrochemical studies of microliter size samples and in studies where the rapid achievement of steady state conditions is desirable. Further, it finds application in conjunction with the electrochemical analysis of non-homogeneous samples. It is to be appreciated, however, that the invention is also applicable to other chemical procedures where precise microdelivery of a reagent is desirable.
I. Routine analysis of the chemical composition of fluids is important in a wide range of fields, including clinical diagnosis, food and drug industries, industrial process control, and environmental studies. Due to the accuracy and reliability that titrimetric methods provide, they are widely used in diagnostic tests.
For accurate results, however, laboratory expertise, relatively large sample volumes, and often devices with expensive micromechanical elements are required for titrimetric studies. In many areas, for example in forensic testing and clinical diagnosis, large quantities of a sample to be studied may be costly or not readily available. To maintain the accuracy of measurements as the size of the sample decreases, the cost of the titration equipment, and the level of skill required, generally increase. Automated addition of reagents further adds to the cost, particularly when delivering microliter size volumes or less.
II. Another analytical technique, the investigation of basic electrochemical reactions, is very important for industrial development in many fields, including semiconductors, the fuel industry, corrosion, quality control, and process monitoring. The rate of electrochemical reactions is limited by the rate of mass transport over the surface of the electrode.
However, the natural processes of diffusion can be accelerated by hydrodynamic electrochemical techniques.
Hydrodynamic electrochemical techniques with enhanced convective mass transport exhibit a number of advantageous voltammetric characteristics. The relative contribution of mass transport limitations with respect to electron kinetics is less pronounced. (Bard, A. J.; Faulkner, L. R.: Electrochemical Methods; John Wiley, (1980)). Steady state conditions (where the current is independent of potential scan direction and time) are attained quickly. Thus, measurements can be carried out with high precision. In addition, at steady state, double layer charging is not a factor.
Traditionally, one of the best methods of obtaining efficient convective mass transport uses a rotating electrode system, such as a rotating disc or ring-disc electrodes. In the latter case, the electrochemically generated species at the disc are swept by laminar flow past the ring, where they can be monitored. Both electrode types have proven to be useful in basic electrochemical studies, such as those of coupled homogeneous reactions (Kissinger, P. T. and Heineman, W. R., Laboratory Techniques in Electroanalytical Chemistry; Marcel Dekker (1984)) and short lived reaction intermediates (Zhao, M. and Scherson, D. A., 64 Anal. Chem. 3064-67 (1992)). Hydrodynamic methods also play an important role in electrochemical preconcentration techniques, such as stripping voltammetry or potentiometry, where enhanced mass transport allows for efficient extraction of the analyte on to the surface of the electrode. Preconcentration of heavy metal trace elements is particularly useful for the analysis of food, environmental, and biological samples, because of the large useful concentration range (1-10-2M), and the simpler, portable and less expensive instrumentation (Bersier, P. M., et al., 119 Analyst 2195-32 (1994)). Potentiometric stripping techniques have been used successfully in the determination of lead in blood samples (Jagner, D., et al., 6 Electroanalysis 285-91 (1994)), in gasoline (Jagner, D., et al., 267 Anal. Chim. Acta. 165-69 (1992)), and of heavy metals in tap water (Jagner, D., et al., 278 Anal. Chim. Acta 237-42 (1993)).
In potentiometric stripping analysis, an oxidizing agent, added to the sample, is used for the stripping of the deposited analyte from the electrode surface. In voltammetric stripping analysis, an anodic voltammetric scan is applied. Potentiometric stripping has advantages over voltammetric stripping in that it is unaffected by dissolved oxygen present in the sample, and does not require sophisticated anodic scanning instrumentation, since the potential is detected in time. (Jagner, D. et al. 278 Anal. Chim. Acta 237-42 (1993)). However, the potentiometric method has a number of disadvantages. For low sample concentrations, the fast stripping rate requires a very high real time data acquisition rate. Also, reproducible hydrodynamic conditions are more important than in anodic stripping, since the driving force of the oxidation is diffusion controlled mass transport.
The detection limit of hydrodynamic techniques can be further reduced by sinusoidally modulating the rotation speed of the electrode (Miller, B. and Bruckenstein, S., 46 Anal. Chem. 2026-33 (1974)).
III. Without techniques for rapid stirring of a test solution, electrochemical transducers, such as simple and modified electrodes, only provide information from the solution layer directly covering, and adjacent to, the sensing surface of the particular electrode used. While optical analytical techniques can produce chemical and other information that reflects bulk solution properties, rather than only surface characteristics, ordinary electrodes are interfacial devices, reflecting only surface characteristics, for example chemical composition at the electrode interface with the solution. Inhomogeneity occurs, for example, when a reagent is introduced to the sample in a nonuniform manner, such as through a membrane in the sample container.
As a consequence, when samples with inhomogeneities are to be analyzed for their average characteristics, electrochemical transducers generally are not suited to making such measurements, unless sufficient stirring of the solution is used to render the sample homogeneous.
For some applications, stirring of the solution is not practical, nor feasible.
IV. The principle of pH-statting (keeping the pH constant) of a sample where an enzyme reaction would otherwise cause the pH to steadily shift was first applied by Knaffl-Lenz in 1923, to establish the rate of an esterase reaction where the enzyme splits an ester into an alcohol and an acid. (Knaffl-Lenz--See Ref. 1) Thus, to keep the pH constant during this process, Knaffl-Lenz kept adding the required amount of base solution to the sample. The rate of addition which ensured an approximately constant pH was used to characterize the rate of the enzyme reaction, i.e. enzyme activity, in the particular experiment. Addition of the base was performed convectively (mechanically), by adding increments of the base solution using the feedback from the actually observed pH.
Today, the same principle is still used for enzyme activity measurements, except that the equipment involved has become more sophisticated. Fully mechanized and automatized instruments are now available that use a pH glass electrode or other method to monitor pH continuously. A feedback control loop (typically a PID controller) ensures that the right amount of acid or base is added at all times. Both analog and digital (computer based) controllers are available and reagent addition can occur in increments or even continuously.
A good description of such a state-of-the-art instrument and its performance and potential applications can be found in "Reaction Kinetics: pH-Stat Analysis with the TitriLab Titration System" (Application Notes, Radiometer--Copenhagen .COPYRGT.1996). Radiometer, Inc. is one of the leading manufacturers of such devices. The range of applications of the technique, however, has become much broader than the original aims of Knaffl-Lenz. The range encompasses determinations in the following areas: 1. Activity of enzymes. 2. Neutralization properties of drugs and other products (e.g. neutralization capacity, and reaction times of antacids). 3. Dissolution rates of minerals and additives for agricultural use (soil chemistry, animal feeds, etc.) 4. Acidity/alkalinity of samples. 5. Biological acid production (bacteria, cells, tissues, etc.). 6. Calcium build-up in muscles.
Many other applications exist or are evolving in research, industry, environmental management and medicine.
The instrumentation, due to the fact that is uses convective (mechanical) addition of an acid or base solution to the samples, requires sensitive and expensive mechanical parts whose fine regulation is complicated. Instruments are, therefore, expensive and require intensive maintenance. Reagent consumption is also high. The typical instrument consists of many different parts that all can malfunction (e.g. autoburette, driving motor and controller, reagent reservoirs, etc.). Another drawback is that relatively large samples are needed, otherwise the mechanical mode of reagent addition may not be fine enough to compensate for the tiny amounts of acid or base produced in a truly small (e.g. 1-20 microliter) sample.
There exists a need for a device for performing analyses on microliter size samples without the requirement for expensive microdelivery systems or extensive laboratory expertise.
Further, there exists a need for hydrodynamic techniques capable of being performed in microliter volumes. The existing techniques discussed all suffer in that they require sample volumes in the mL range. Constructing a rotating electrode of the smaller dimensions required for microliter sample volumes is not economically or mechanically feasible. Reproducibility of hydrodynamic conditions also becomes more difficult for smaller sample volumes.
Further, there exists a need for electrochemical techniques capable of analyzing a non-homogeneous sample and providing an average measurement corresponding to the solution as a whole, without mixing the sample.
Finally, there exists a need for pH-statting of microliter sized samples.
The present invention provides a new and improved apparatus and method for studies and analyses of microliter size samples which overcomes the above referenced problems and others.